One of the problems in the treatment of allergic diseases is the lack of diagnostic techniques which are sufficiently specific and sensitive and which do not imply any risk to the patient. Furthermore, there is a need for a rapid, simple, and inexpensive diagnostic technique which would ensure a decrease in the number of patients now waiting for adequate treatment.
In human blood and tissue there exist specific cells (basophil leucocytes and mast cells) which are involved in the allergic reaction. On the surface of these cells is a distinct class of antibodies (IgE-antibodies). When e.g. a patient is allergic to cat, an allergic reaction is caused by the antibodies on the surface of these cells which specifically recognize the protein structure of cat dandruff. When inhaled, cat protein comes in contact with the mast cells and the basophil leucocytes of the lung tissue. The antibodies on the cell surface will react with cat protein, thereby triggering a reaction in the cell, which in turn causes the release of a number of substances (allergic mediators). These allergic mediators are responsible for the symptoms of the patient.
For many years it has been possible to imitate the allergic reaction in vitro. This is done by exposing a blood sample from the allergic patient to e.g. cat protein. Allergic mediators are released from the basophil leucocytes in the blood sample. Of these mediators it is possible to determine one substance, i.e. histamine. Therefore, if the patient is allergic to cat, it is possible to determine the release of histamine from the cells. Methods based on this principle represent the best assay for obtaining a correct diagnosis, and are explained in detail below.
By means of histamine determination it is possible to diagnose the responsible antigens (e.g. grass pollen, animal dandruff, drugs, foodstuffs, mold fungi, and bacteria) in patients with allergic diseases (asthma, urticaria, and hay fever).
A general problem of this test is that it is difficult to perform in the laboratory. Few tests can be performed daily and the demand for laboratory technicians is high. Therefore, a simplification of the test is greatly needed so that it could be introduced in the daily diagnostic routine of the clinic.
As mentioned, a number of assays exist for detecting histamine in liquids. Thus, the original histamine determination was described by Shore et al. and was based on a fluorometric assay (ref. 1).
The principle of this assay described in Hoppe-Seyler's 2. Physiol. Chem. 353: 911-920, 1972, is a coupling of histamine to a fluorophore (o-phthalaldehyde), whereby a ring structure is formed. The concentration of this ring structure, which can be determined spectrophotometrically, depends on the amount of histamine present. The method was later been modified to increase specificity and sensitivity. Stahl Skov & Norn, Acta Allergol. 32: 170-182, 1977, (ref. 2) have thus simplified the assay by allergen-provocation of Ficoll-Hypaque-isolated cell suspensions containing 0.5-2% basophil leucocytes instead of whole blood. By fractionation of the blood, interfering substances are removed, so that the histamine content of the basophil leucocytes can be determined directly, avoiding a long extraction procedure. However, the fractionation procedure (gradient centrifugation) necessary to remove interfering substances is time consuming and difficult to perform, and Siraganian therefore developed an autoanalytical fluorometric method Int. Archs Allergy appl. Immun. 49: 108-110, 1975. This method is used in the clinic, but has only found limited application, due to its demand for technical experience, constant monitoring, and expensive apparatus.
Taylor et al. Int. Archs Allergy appl. Immun. 61: 19-27, 1980 has developed another assay for determining histamine in biological material. This very sensitive and specific method is an enzymatic isotope technique based on methylation of histamine by means of N-methyltransferase. This assay is useful in determining small amounts of histamine in tissue, blood, and urine. However, the need for a routine method to be used in the clinic is not satisfied by this method since the number of assays that can be performed in a day is low (approx. 30) and the assay time is long.
Stahl Skov et al. Allergy 34: 261-263, 1979, have developed another method based on in vitro incorporation of radioactively labelled histamine in the basophil cells of the patient, where the release of labelled histamine is determined after provocation with the suspected allergens.
However, as illustrated below, a poor correlation with the release of histamine determined fluorometrically was obtained by this method.